The device was imaged under different amplitudes and frequencies to verify the ability of the actuator to deliver the correct travel distances. Likewise, the ability of 17-AAG side effects the actuator to be consistent over time was measured by comparing the initial and final cycles of a three hour long stretch. Cell culture and Gelfoam seeding The protocol was approved by the Boston University IACUC. Neonatal rat pulmonary fibroblasts (NNRLF) were isolated from 2�C3 d old Sprague-Dawley rats and were cultured in Dulbecco��s modified Eagle’s medium (DMEM) with 5% fetal bovine serum (FBS), 1% penicillin/streptomycin antibiotic cocktail, 1% sodium pyruvate and 1% nonessential amino acids. The cells were cultured in flasks for one week before being seeded on Gelfoam constructs.

Gelfoam constructs (Pharmacia and Upjohn) were cut into 4 cm �� 1 cm �� 0.3 cm pieces the day before seeding and were equilibrated in NNRLF medium overnight prior to seeding. The constructs were seeded by spot pipetting 250,000 cells over the entire 4 cm �� 1 cm surface. Samples were then placed in an incubator for 2�C3 h and then into centrifuge tubes containing NNRLF medium. The medium was changed 24 h after seeding and at 3 d intervals thereafter. The constructs were cultured for 8 d before stretching. Stretching protocol Gelfoam constructs were stretched on the 8th day following seeding without changing the medium. Samples were attached to the device using the special clamps and the stretching was performed on six samples simultaneously. Two unstretched constructs were maintained as controls for each day.

The two types of stimuli delivered were monotonous cyclic and variable cyclic stretch. Variable stretch delivers a sequence of sinusoids in which each cycle has a different strain amplitude (��) and frequency (f). Here we defined the variability of the stretch signal to be an interval described by the percentage of the mean strain; thus, a signal with a mean strain of 20% and an imposed variability of 25% would deliver peak strains in each cycle in the range of 15 to 25% with equal probability. The corresponding strain and frequency were fixed by the equation ��f = C, such that a larger peak strain would have a smaller frequency resulting in a constant strain rate.All stretched conditions received an average strain amplitude of 20% at an average frequency of 0.2 Hz.

In total, there were four conditions: (1) monotonous stretch, which corresponds to 0% variability, and consists of a single repeated sinusoidal stretch at 20% strain (2) 25% variability, (3) 50% variability and (4) 75% variability. All stretches were applied for three hours. Following stretching, the samples were removed from the wells and cut from the clamps. Only the portion of the sample that experienced stretch was collected for analysis. mRNA expression All Gelfoam samples were collected for total Cilengitide RNA purification immediately following stretching.